The present invention relates to haemostasis.
Haemostasis is a tightly regulated process which causes bleeding to stop. In the body, circulating blood remains fluid under normal conditions, but forms localized clots when the integrity of the vascular system is breeched. Trauma, infection, and inflammation all activate the blood's clotting system, which depends on the interaction of two separate systems: enzymatic proteins in a clotting cascade (e.g., clotting factors such as Factor VII or Factor IX) and activated platelets. The two systems work in concert to plug defects in the broken vessels.
A blood clot (also called a thrombus) that forms during haemostasis is made of two parts—a platelet plug and a mesh of cross-linked fibrin protein. The fibrin results from cleavage of fibrinogen into fibrin by thrombin which is activated during the clotting cascade (see FIG. 1). A blood clot needs to be of sufficient strength to resist dislodgement by circulating blood or mechanical movement. If a particular clotting factor is dysfunctional or absent, as in hemophilia, an insufficient amount of fibrin forms. Similarly, massive consumption of clotting factors in a trauma situation decreases the amount of fibrin formed. Inadequate numbers of platelets resulting from trauma, surgery, or chemotherapy also decrease platelet aggregation, as do genetic disorders, uremia, or salicylate therapy. Ultimately, reduced fibrin formation or platelet aggregation results in clots of inadequate tensile strength. This hypocoagulable state makes the patient prone to bleeding. Conversely, endothelial injury, stasis, cancer, genetic diseases, or other hypercoagulable states lead to thrombosis (i.e., blood clot) formation, exemplified in deep-vein thromboses, pulmonary emboli, and arterial occlusions such as stroke and myocardial infarction.
The precursor of plasmin, plasminogen, is an inactive protein that is incorporated into a blood clot. Tissue plasminogen activator (t-PA) and urokinase are able to convert plasminogen to plasmin, thus activating it and allowing fibrinolysis to occur. Fibrinolysis, the process of breaking down blood clots, so that they do not become problematic, is a normal biological process. Normally, t-PA is released very slowly into the blood by the damaged endothelium of blood vessels. As a result, after bleeding is stopped, the clot is broken down as the inactive plasminogen in the clot is activated to become plasmin, which acts to break down the fibrin mesh holding the clot together. The resulting fragments, called fibrin degradation products (FDPs), are then cleared by other enzymes, or by the kidney and liver.
In some situations, hyperfibrinolysis can also occur. This condition, a form of coagulopathy (bleeding disorder) with markedly enhanced fibrinolytic activity, results in increased and sometimes fatal bleeding.
Hyperfibrinolysis can be acquired or can be congenital. Congenital reasons for hyperfibrinolysis are rare and include deficiency of alpha-2-antiplasmin (alpha-2-plasmin inhibitor) and deficiency in plasminogen activator inhibitor type 1 (PAI-1). The affected individuals show a hemophilia-like bleeding phenotype.
Acquired hyperfibrinolysis can occur in patient with liver disease, patients with severe trauma, patients undergoing major surgical procedures, and patients with other conditions. Indeed, up to 20% of severely injured trauma patients are affected by acquired hyperfibrinolysis, as are other patients with massive hemorrhage.
Known methods to detect fibrinolysis and hyperfibrinolysis include indirect immunochemical methods which detect the elevation of biomarkers such as D-Dimer (cross-linked fibrin degradation products), fibrinogen split products (FSP), complexes of plasmin and alpha-2-antiplasmin (PAP). However, the sensitivity and specificity of these methods is limited because elevation of these biomarkers can also occur induced in other conditions. The classical coagulations tests such as PT (prothrombin time), aPPT (activated partial thromboplasin time) or thrombin time are not very sensitive for fibrinolysis and hyperfibrinolysis, and are influenced by numerous other variables.
Thus, there is a need to for methods to rapidly and accurately diagnose and/or detect fibrinolysis and hyperfibrinolysis.